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BET protein Brd4 activates transcription in neurons and BET inhibitor Jq1 blocks memory in mice

Nature Neuroscience volume 18, pages 1464–1473 (2015)Cite this article

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Abstract

Precise regulation of transcription is crucial for the cellular mechanisms underlying memory formation. However, the link between neuronal stimulation and the proteins that directly interact with histone modifications to activate transcription in neurons remains unclear. Brd4 is a member of the bromodomain and extra-terminal domain (BET) protein family, which binds acetylated histones and is a critical regulator of transcription in many cell types, including transcription in response to external cues. Small molecule BET inhibitors are in clinical trials, yet almost nothing is known about Brd4 function in the brain. Here we show that Brd4 mediates the transcriptional regulation underlying learning and memory. The loss of Brd4 function affects critical synaptic proteins, which results in memory deficits in mice but also decreases seizure susceptibility. Thus Brd4 provides a critical link between neuronal activation and the transcriptional responses that occur during memory formation.

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Figure 1: Brd4 is expressed in neurons throughout the brain.
Figure 2: Brd4 regulates IEG transcription in response to stimulation in neurons.
Figure 3: Genome-wide analysis of effects of Jq1.
Figure 4: CK2 regulates Brd4 in neurons.
Figure 5: Phosphorylation of Brd4 is critical for its function.
Figure 6: Brd4 regulates synaptic receptor subunit GluA1.
Figure 7: Jq1 affects mouse behavior.
Figure 8: Jq1 decreases seizure susceptibility.

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Acknowledgements

We thank K. Ozato for the Brd4 construct, J. Gresack and the Rockefeller behavioral core for guidance and suggestions, K. Thomas for microscopy expertise, the Memorial Sloan-Kettering Cancer Center histology core for immunohistochemistry support, A. Soshnev for the schematic model graphic, J. Gerace for editing and members of the Allis laboratory for feedback. Support for this work was provided by a grant from the Robertson Foundation, a Ruth Kirschstein US National Research Service Award fellowship (F32MH103921), the US National Institutes of Health (R01 NS34389, NS081706) and a Simons Foundation Research Award. R.B.D. is an Investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University, New York, New York, USA

    Erica Korb & C David Allis

  2. Laboratory of Molecular Neuro-oncology, The Rockefeller University, New York, New York, USA

    Margo Herre, Ilana Zucker-Scharff & Robert B Darnell

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  1. Erica Korb
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  2. Margo Herre
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Contributions

E.K. wrote the manuscript and designed and carried out experiments. M.H. helped carry out behavioral testing and molecular studies. I.Z.-S. helped with seizure testing. R.B.D. helped initiate and design the project and provided feedback, and C.D.A. provided support, feedback and guidance.

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Correspondence to Erica Korb or C David Allis.

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Integrated supplementary information

Supplementary Figure 1 Brd4 is expressed in the brain.

(a) Whole cell lysate of regions of adult mouse brain. Representative blot of 3 biological replicates. (b, c) Cultured cortical neurons stained with Brd4 and CamKII (b) or Gaba (c). (d) Brd4 mRNA from cultured neurons after BDNF treatment (one sample t-test for 0.5 hours n = 14, for 2 hours n = 13, for 4 hours n = 14, for 8 hours n = 12, for 24 hours n = 13, P = 0.0339, t = 2.01). (e, f) Brd4 protein after short (e) or long (f) BDNF treatment. Representative blot of 3 biological replicates. *, p<0.05. Full-length blots are presented in Supplementary Figure 10. min, minutes. h, hours. Error bars represent standard error. Scale bar is 10 μm.

Supplementary Figure 2 Brd4 regulates IEG transcription in response neurons.

(a, b, c) Time course of the BDNF-induced increase in Arc (a), Fos (b) and Nr4a1 (c) after (-) or (+)Jq1 treatment. (d) Brd4 mRNA 3 days after lentiviral infection of an siRNA targeted against Brd4 compared to a scrambled siRNA (one-sample t test, n = 7 biological replicates P = 6.18E-6, t = 13.07). (e) BDNF-induced MAPK phosphorylation after Brd4 knockdown with lentiviral infection. Representative of 2 biological replicates. (f, g) Staining (f) and quantification (g) of Brd4 in neurons 5 days after transfection with GFP and Brd4 siRNA (unpaired two-sided t test n = 45 neurons for control siRNA and 37 neurons for Brd4 siRNA from 5 biological replicates, P = 6.38E-16, t = 10.093). (h, i) Brd2 (h) and Brd3 (i) mRNA fold decrease 5 days after transfection in N2A cells (one sample t-test n = 3 biological replicates, for Brd2 P = 0.0157, t = 5.51, for Brd3 P = 0.019, t = 4.92). (i) Quantification of Brd4 staining 5 days after transfection with different siRNAs targeted against Brd4. N = 47 neurons for control siRNA (upaired two-tailed t test for control n = 47 for Brd4 siRNA1 n = 43 and for Brd4 siRNA2 n = 37 neurons from 2 biological replicates, for siRNA1 P = 2.24E-38, t = 22.91, for siRNA2 P = 2.82E-25, t = 15.18). (k, l) Staining (k) and quantification (l) of Arc with or without 30 minutes BDNF stimulation after transfection with GFP and either a nontargeting siRNA or different Brd4 siRNA constructs (upaired two-tailed t test for control n = 25, for control + BDNF n = 23, for Brd4 siRNA1 n = 22, for Brd4 siRNA1 + BDNF n = 22, for Brd4 siRNA2 n = 14, and for Brd4 siRNA2 + BDNF n = 26 from 2 biological replicates, for control vs BDNF P = 9.53E-4, t = 3.432). (m) Quantification of Arc staining in neurons transfected with a long or short form of Brd4 (upaired two-tailed t test for control n = 42 neurons, for long Brd4 n = 26 neurons and for short Brd4 n = 30 neurons, for control vs long Brd4 P = 0.015, t = 2.48, for long vs short Brd4 P = 0.0016, t = 3.29). (n) Decrease in Arc, Fos and Nr4a1 RNA after 24 hours of Jq1 treatment in the absence of exogenous stimulation in cultured cortical neurons (one sample t test, for Arc n = 13 biological replicates P = 3.098E-5, t = 6.58, for Fos n = 13 biological replicates P = 0.013, t = 2.65, and for Nr4a1 n = 11, P = 0.00104, t = 4.75). (o) Jq1 does not affect global H3 or H4 acetyl levels. (p) MAPK phosphorylation in response to BDNF with or without Jq1 pretreatment. Representative blot of 2 biological replicates. a.u., arbitrary units. #, p< 0.05 for univariate analysis. ###, p< 0.001 for univariate analysis. *, p< 0.05. ***, p<0.001. Full-length blots are presented in Supplementary Figure 10. Error bars represent standard error. Scale bar is 10 μM.

Supplementary Figure 3 RNA-sequencing analysis of effects of Jq1 in neurons.

(a) Experimental paradigm for genome-wide analysis of effects of Jq1. (b) Heat map of fold induction of all genes significantly induced by BDNF after treatment with (-) or (+) Jq1. (c) Top GO terms of genes clusters enriched in Jq1 up-regulated genes. Data represents averages of 3 biological replicates. (d) Histone acetylation at promoter regions of IEGs in response to BDNF for acetyl marks that recruit Brd4. N = 3 biological replicates. (e) Western blot analysis of acetylated histone H4K16 and H3K14 with (-) or (+) Jq1. Representative blot of 3 biological replicates. Full-length blots are presented in Supplementary Figure 11. min, minutes.

Supplementary Figure 4 CK2 regulates Brd4 in neurons.

(a, b, c) Brd4 ChIP-qPCR analysis in neurons stimulated with BDNF to measure Brd4 at promoters of Arc (a), Fos (b), and Nr4a1 (c). (d, e, f) Brd2 ChIP in neurons stimulated with BDNF at promoters of Arc (d), Fos (e), and Nr4a1 (f). N = 3 biological replicates. (g, h, i) Brd3 ChIP in neurons stimulated with BDNF at promoters of Arc (g), Fos (h), and Nr4a1 (i). N = 3 biological replicates for a-i. (j) MAPK phosphorylation in response to BDNF in the presence of TBB. Representative of 3 biological replicates. (k, l, m) CBP ChIP-qPCR analysis in neurons stimulated with BDNF with Jq1 or TBB pretreatment to measure Brd4 at promoters of Arc (k), Fos (l), and Nr4a1 (m). N = 2 biological replicates. (n) Quantification of Ck2 expression in neurons transfected with GFP and either nontargeting siRNA, or siRNA targeting CK2 (unpaired two-tailed t test, for control siRNA n = 24 neurons and for CK2 siRNA n = 26 neurons from 3 biological replicates, P = 8.05E-5, t = 3.29). (o, p) Arc and CK2 staining (o) and Arc quantification (p) after a 30 minute BDNF stimulation of neurons transfected with GFP and either a nontargeting siRNA pool or a siRNA pool targeting CK2 (unpaired two-tailed t test, for control siRNA n = 50 neurons, n = 39 for BDNF, for CK2 siRNA n = 39, and for CK2 siRNA + BDNF n = 37 from 5 biological replicates, control vs BDNF, P = 1.21E-5, t = 5.244, for BDNF vs CK2 siRNA + BDNF P = 7.09E-7, t = 5.42). (q) Mobile fraction quantification of EGFP-Brd4 (unpaired two-tailed t test for control n = 15 for BDNF 18 neurons. P = 5.389E-5, t = 4.73). ***, p<0.001. a.u. arbitrary units. a.u. arbitrary units. min, minutes. Error bars represent standard error. Full-length blots are presented in Supplementary Figure 11. Scale bar is 10 μm.

Supplementary Figure 5 Phosphorylation of the CK2 site in Brd4.

(a) Dot blot for phopho-Brd4 antisera using target and control peptides shows specific binding to peptides containing phosphorylated S492. (b) Western blot with phopho-Brd4 antisera in lyates treated with phosphatase or control lysates. Blots are representative of 3 replicates. (c) Quantification of Brd4 expression shows elevated and equivalent Brd4 levels in neurons transfected with GFP and Brd4 with deletions or mutations in the CK2 site (unpaired two-sided t test, for GFP n = 68, for Brd4 n = 61, P = 4.596E-17, t = 9.73, for CK2 deletion n = 46, P = 1.26E-27, t = 14.57 for deletion 492-494 n = 44, P = 1.48E-22, t = 12.37, for S492A n = 54, P = 4.14E-34, t = 17.17, for SSS492ESE n = 51 from 5 biological replicates, P = 7.82E-24, t = 12.73, no significant differences between Brd4 constructs). (d) Example images of EGFP-Brd4 with CK2 site mutations costained with H4K16acetyl. (e) Example graphs showing corresponding changes in fluorescent signal of Brd4 and H4K16acetyl in sections of neuronal nuclei. **, p<0.01. ***, p<0.001. Full-length blots are presented in Supplementary Figure 11. Error bars represent standard error. Scale bar is 5 μm.

Supplementary Figure 6 Brd4 inhibition decreases surface GluA1.

(a) Quantification of mRNA after different times of Jq1 treatment. N = 3 biological replicates for Arc and Fos, N = 8 for Nr4a1, N = 9 for Gria1, N = 4 for Bdnf and N = 5 for Brd4. (b) Quantification of hippocampal neurons treated with Jq1 for 24 hours and stained for surface GluA1 (unpaired two-sided t test, for control n = 13 neurons and for Jq1 n = 12 neurons from 2 biological replicates, P = 0.0015, t = 3.56). (c, d) Images (c) and quantification (d) of surface GluA1 staining 5 days after transfection with different siRNAs targeted against Brd4 (unpaired two-sided t test, for control siRNA n = 14 neurons, for Brd4 siRNA1 n = 13 neurons, and for Brd4 siRNA2 n = 14 neurons from 2 biological replicates, for control vs siRNA1 P = 0.0114, t = 2.61, for control vs siRNA2 P = 0.0078, t = 2.76). (e) Quantification of spine number in neurons treated with Jq1 for 24 hours (unpaired two-sided t test, n = 68 neurons for control for Jq1 and n = 66 from 2 biological replicates). (f) Quantification of staining of surface GluA1 in neurons with long or short forms of Brd4 (unpaired two-sided t test, for control n = 15 neurons, for long Brd4 n = 17, and for short Brd4 n = 18 from 2 biological replicates, for control vs long Brd4 P = 0.0161, t = 2.55, for long vs short Brd4 P = 0.0308, t = 2.26). (g) ChIP for Brd4 at the gria1 promoter region. N = 7 biological replicates for control conditions, 3 biological replicates for Jq1 conditions. (h) ChIPs for histone acetylation at the gria1 promoter region. N = 2 biological replicates. *, p<0.05. ***, p<0.001. a.u. arbitrary units. n.s., non-significant. Scale bar is 10 μm.

Supplementary Figure 7 Jq1 affects mouse behavior.

(a) Weight change in mice treated daily with Jq1 or vehicle. (b) Zone preference ratio in an open field in mice treated with vehicle or with Jq1 for 1 or 3 weeks. (c) Total distance traveled during 1 hour of an open field in mice treated with vehicle or with Jq1 for 1 or 3 weeks. (d) Time spent moving during 1 hour of an open field in mice treated with vehicle or with Jq1 for 1 or 3 weeks. (e) Distance traveled in the novel object box during habituation. (f-i) Time spent exploring objects for memory tests during initial exposure (f) or during testing (g) for memory tests or for learning tests during initial exposure (h) or during testing (i). (j) Discrimination index of time spent with a novel vs familiar object when mice are tested immediately after initial exposure to objects (for learning tests, n = 10 mice for DMSO and 9 mice for 1 week Jq1. No significant difference between DMSO and Jq1, for one sample t test for DMSO P = 0.00325, t = 4.14, for Jq1 P = 0.00103, t = 5.017). (k) Percent of time spent freezing in response to a tone before training, during training, or 1 day after training in a new context (2-way ANOVA with posthoc t test, P = 2E-16, F = 165.7, df = 130) (l) Percent of time spent freezing before, immediately after, or 1 day after fear conditioning training in the training context or in a novel context. (2-way ANOVA with posthoc t test, P = 2E-16, F = 80.72, df = 110). N = 10 mice for control and 3 week Jq1 and 9 mice for 1 week Jq1 for testing groups. *, p<0.05. ***, p<0.001. n.s., nonsignificant. Error bars represent standard error.

Supplementary Figure 8 Jq1 affects seizure susceptibility.

(a) Quantification of mRNA from cortical tissue from mice after behavioral testing following treatment with DMSO or 1 week of Jq1 (unpaired two-sided t test for dmso n = 9 brains and for Jq1 n = 8, for gria1 P = 0.00517, t value = 3.27, for Nr4a1 P = 0.018, t value = 2.64). (b) Western of GluA1 levels after Jq1 treatment. (c) Data for each level of Racine scoring of seizure induction for mice treated for one week with either DMSO or Jq1 and then given pentylenetetrazol (PTZ) to induce seizures. (d) Percent mortality of male mice after seizure injection by PTZ. (e) Seizure susceptibility score of female mice treated for one week with either DMSO or Jq1 and given pentylenetetrazol (PTZ) to induce seizures. (f) Percent mortality of female mice after seizure injection by PTZ. (g, h) Seizure susceptibility scores during kindling for individual mice treated with DMSO (f) or Jq1 (g). N = 9 mice per group. *, p<0.05. ***, p<0.001. Error bars represent standard error. Full-length blots are presented in Supplementary Figure 11.

Supplementary Figure 9 Model of Brd4 function in neurons.

Neuronal activation by signaling molecules such as BDNF trigger activation of CK2 which in turn phosphorylates Brd4. Brd4 binds to acetylated lysines on histone proteins and activates expression of neuronal genes including IEGs which then affect synaptic proteins. Inhibition of Brd4 by Jq1 affects aspects synaptic function and memory formation.

Supplementary Figure 10 Original western blots for Figure 1 and Supplementary Figures 1 and 2.

Supplementary Figure 11 Original western blots for Figures 3, 4, 5, 6 and Supplementary Figures 4, 5 and 8.

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Korb, E., Herre, M., Zucker-Scharff, I. et al. BET protein Brd4 activates transcription in neurons and BET inhibitor Jq1 blocks memory in mice. Nat Neurosci 18, 1464–1473 (2015). https://doi.org/10.1038/nn.4095

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